Many cells exhibit calcium spikes (periodic transient increases in cytosolic CA2+) when stimulated by a neurotransmitter, hormone, or growth factor. The overall goal of this research is to elucidate the molecular mechanism of calcium spiking and delineate its role in signal transduction. The rat basophilic leukemic cell (RBL cell) will be studied as a model secretory cell, the rat hepatocyte as an integrator of diverse stimuli, and the PC12 cell as a model neuron. Calcium spiking will be monitored by photon-counting fluorescence microscopy of single cells containing an indicator such as fluo-3. Biochemical, fluorescence, and ultrastructural studies of intact cells, permeabilized cells, purified proteins, and reconstituted membrane assemblies will be carried out to answer: (1) How are calcium spikes generated? We will test a molecular model for calcium spiking that is based on four elements: cooperativity and positive feedback between inositol 1,4,5- trisphosphate (IP3) and cytosolic Ca2+, delayed deactivation by mitochondrial uptake of Ca2+, and reactivation by refilling of the endoplasmic reticulum Ca2+ store. Specific inhibitors will pinpoint the contributions of particular processes to spike generation. (2) How is calcium spiking modulated? The effects of calcium influx into the cell, intracellular pH, the level of phosphoinositides, and phosphorylation state of components of the spike generator will be determined. (3) How is spiking altered by the interplay of the phosphoinositide cascade with other signal transduction pathways? The modulatory actions of the cyclic AmP cascade, growth factors, voltage-sensitive calcium channels, and lithium ion will be investigated. (4) How do calcium spikes trigger effector events such as exocytosis and memory? The distribution of F- actin and myosin in stimulated RBL cells will be determined by fluorescence and immunoelectron microscopy to determine whether individual spikes induce discrete cytoskeletal rearrangements and to relate them to granule release. The Ca2+/calmodulin-dependent protein kinase of PC12 cells will be studied as a model memory protein. We will measure how calcium spikes switch this protein between different functional states as expressed by their degree of phosphorylation- autophosphorylation activity, and kinase activity for exogenous substrates such as tyrosine hydroxylase. A deeper understanding of calcium spiking is likely to reveal how digital logic is used within cells to process information and achieve timing control (as in circadian rhythms). Some neuropsychiatric disorders may arise from kinetic mismatches of components of the spike generator. Information about spiking should lead to a better understanding of the therapeutic action of lithium ion in manic-depressive disorders.
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